Many different techniques are currently used to identify nucleic acid sequences of deoxyribonucleic acids (DNA). Nick translation with E. coli polymerase I or polymerization with T4 DNA polymerase produces complementary DNA strands. By these approaches, hybridization probes, specific nucleic acid stretches which can be analytically identified, are formed. These hybridization probes can be used to identify some target DNA and RNA. This permits a scientist to detect the presence of specific DNA or RNA sequences in a genome.
There are different types of hybridization probes. By far the most commonly used hybridization probes contain radioactive DNA building blocks, wherein one of the atoms are radioactive and identifiable by radioactive assay methods. These radioactive DNA building blocks form excellent hybridization probes because they act in the same manner as a naturally occurring nucleic acid. In other words, the Pol I enzyme cannot distinguish the radioactive DNA building block from a non-radioactive one. The radioactively labeled nucleic acid stretches which contain the radioactive DNA building blocks are detected by X-ray film.
Radioactive hybridization probes present three distinct problems. The detection method for these probes involves multiple separation steps and cannot be conducted in solution preventing automation of genetic screening. More significantly, because of a much greater concern with the health and environmental hazards posed by these radioactive probes, appropriate safe handling of the hybridization probes is becoming more expensive and difficult. Finally, .sup.32 P radiolabeled DNA probes are unstable and have a limited shelf life.
There are alternatives to radioactive hybridization probes. Probes which can be detected colorometrically are known. For example, Langer, in Proceedings of the National Academy of Science, Vol. 78, No. 11, pp. 6633-6637, Nov. 1981, discloses a biotin labeled nucleic acid. Specifically, Langer et al. report the production of a biotin labeled uridine triphosphate as well as a biotin labeled deoxyuridine triphosphate. Both of these building blocks are labeled with biotin at the C-5 position of the pyrimidine ring using an allylamine linker arm. The biotin labeled deoxyuridylic acid can be then incorporated into DNA using E. coli polymerase I. These hybridization probes are visualized by colorimetric assays.
There are problems with the colorimetric identification of biotin labeled DNA. Although kits have been made available by Enzo Biochem, Inc., New York or Bethesda Research Laboratories, Maryland, the required multiple steps for identification of biotin labeled DNA make it necessary to use specifically trained personnel. In addition, the color generating process that uses an enzyme to catalyze the synthesis of a colored or fluorescent molecule requires many hours for the visualization of the labeled DNA.
A number of nitroxide labeled nucleic acids have been reported, for example, Bobst et al in Journal of Molecular Biology (1984), 173, 63-74, reported various nitroxide labeled deoxyuridines wherein the nitroxide group was attached to the C-5 position of the pyrimidine ring. The structures reported are set forth below. ##STR1##
Further, these nitroxide labeled nucleic acids can be identified by electron spin resonance spectroscopy. Such a method is advantageous since it can be conducted using solution chemistry which greatly simplifies the identification process. Bobst et al also determined that these nitroxide labeled nucleic acids could be incorporated into nucleic acid homopolymers using terminal transferase. However, they were not taken up by the Pol I enzyme in nick translation experiments or by the T4 DNA polymerase with double stranded DNA as a template. Therefore these are totally unsuitable for use in formation of hybridization probes using template specific enzymes.